Pheromone Biosynthesis Research Articles

Identification and expression of PBAN/diapause hormone and GPCRs from Aedes aegypti

Neuropeptides control various physiological functions and constitute more than 90% of insect hormones. The pheromone biosynthesis activating neuropeptide (PBAN)/pyrokinin family is a major group of insect neuropeptides and is well conserved in Insecta. This family of peptides has at least two closely related G-protein-coupled receptors (GPCRs) activated by PBAN and a diapause hormone (DH). They have been shown to control several biological activities including pheromone production and diapause induction in moths. However, beyond some moth species, the biological function(s) of PBAN/pyrokinin peptides are largely unknown although these peptides are found in all insects. In this study we identified and characterized PBAN/pyrokinin peptides and corresponding GPCRs from the mosquito, Aedes aegypti. Ae. aegypti PBAN mRNA encodes four putative peptides including PBAN and DH, and is expressed in females and males during all life stages. The PBAN receptor (PBAN-R) and the DH receptor (DH-R) were functionally expressed and confirmed through binding assays with PBAN and DH peptides. These receptors are differentially expressed from eggs to adults with the relative gene expression of the PBAN-R significantly lower during the 4th instar larval (L4) and pupal (P1–P2) stages compared to the 2nd and 3rd instar larval stages (L2 and L3). However, DH-R expression level is consistently 4–10 times higher than the PBAN-R in the same period, suggesting that PBAN-R is downregulated in the late larval and pupal stages, whereas DH-R stays upregulated throughout all developmental stages. PBAN/pyrokinin mRNA expression remains high in all stages since it produces PBAN and DH peptides. This study provides the foundation for determining the function(s) of the PBAN/pyrokinin peptides in mosquitoes and establishes data critical to the development of methods for disruption of these hormone actions as a novel strategy for mosquito control.

Biosynthesis and PBAN-Regulated Transport of Pheromone Polyenes in the Winter Moth, Operophtera brumata

The trienoic and tetraenoic polyenes, (3Z,6Z,9Z)-3,6,9-nonadecatriene, (3Z,6Z,9Z)-3,6,9-henicosatriene, and (3Z,6Z,9Z)-1,3,6,9-henicosatetraene were found in the abdominal cuticle and pheromone gland of the winter moth Operophtera brumata L. (Lepidoptera: Geometridae), in addition to the previously identified single component sex pheromone (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene. The pheromone biosynthesis activating neuropeptide (PBAN) is involved in the regulation of polyene transport from abdominal cuticle to the pheromone gland. In vivo deuterium labeling experiments showed that (11Z,14Z,17Z)-11,14,17-icosatrienoic acid, the malonate elongation product of linolenic acid, (9Z,12Z,15Z)-9,12,15-octadecatrienoic acid, is used to produce (3Z,6Z,9Z)-3,6,9-nonadecatriene and (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene.

Transcriptome exploration of the sex pheromone gland of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae).

BackgroundMolecules involved in pheromone biosynthesis may represent alternative targets for insect population control. This may be particularly useful in managing the reproduction of Lutzomyia longipalpis, the main vector of the protozoan parasite Leishmania infantum in Latin America. Besides the chemical identity of the major components of the L. longipalpis sex pheromone, there is no information regarding the molecular biology behind its production. To understand this process, obtaining information on which genes are expressed in the pheromone gland is essential.MethodsIn this study we used a transcriptomic approach to explore the pheromone gland and adjacent abdominal tergites in order to obtain substantial general sequence information. We used a laboratory-reared L. longipalpis (one spot, 9-Methyl GermacreneB) population, captured in Lapinha Cave, state of Minas Gerais, Brazil for this analysis.ResultsFrom a total of 3,547 cDNA clones, 2,502 high quality sequences from the pheromone gland and adjacent tissues were obtained and assembled into 1,387 contigs. Through blast searches of public databases, a group of transcripts encoding proteins potentially involved in the production of terpenoid precursors were identified in the 4th abdominal tergite, the segment containing the pheromone gland. Among them, protein-coding transcripts for four enzymes of the mevalonate pathway such as 3-hydroxyl-3-methyl glutaryl CoA reductase, phosphomevalonate kinase, diphosphomevalonate descarboxylase, and isopentenyl pyrophosphate isomerase were identified. Moreover, transcripts coding for farnesyl diphosphate synthase and NADP+ dependent farnesol dehydrogenase were also found in the same tergite. Additionally, genes potentially involved in pheromone transportation were identified from the three abdominal tergites analyzed.ConclusionThis study constitutes the first transcriptomic analysis exploring the repertoire of genes expressed in the tissue containing the L. longipalpis pheromone gland as well as the flanking tissues. Using a comparative approach, a set of molecules potentially present in the mevalonate pathway emerge as interesting subjects for further study regarding their association to pheromone biosynthesis. The sequences presented here may be used as a reference set for future research on pheromone production or other characteristics of pheromone communication in this insect. Moreover, some matches for transcripts of unknown function may provide fertile ground of an in-depth study of pheromone-gland specific molecules.

Development of a Method to Quantitate Nematode Pheromone for Study of Small-Molecule Metabolism in Caenorhabditis elegans

Pheromones produced by Caenorhabditis elegans are considered key regulators of development, mating, and social behaviors in this organism. Here, we present a rapid mass spectrometry-based method (PheroQu) for absolute quantitation of nematode pheromones (e.g., daumone 1, 2, and 3) both in C. elegans worm bodies (as few as 20 worms) and in liquid culture medium. Pheromones were separated by ultra performance liquid chromatography and monitored by a positive electrospray ionization detector in the multiple-reaction monitoring mode. The daf-22 mutant worms were used as surrogate matrix for calibration, and stable deuterated isotope-containing pheromone was used as internal standard for measuring changes in pheromones in N2 wild-type and other strains under different growth conditions. The worm-body pheromones were extracted by acidified acetonitrile solvent, and the secreted pheromones were extracted from culture medium with solid-phase extraction cartridges. The run time was achieved in less than 2 min. The method was validated for specificity, linearity, accuracy, precision, recovery, and stability. The assay was linear over an amount range of 2-250 fmol, and the limit of quantitation was 2 fmol amounts for daumone 1, 2, and 3 in both worm bodies and culture medium. With the PheroQu method, we were able to identify the location of pheromone biosynthesis and determine the changes in different pheromone types synthesized, according to developmental stages and aging process. This method, which is simple, rapid, sensitive, and specific, will be useful for the study of small-molecule metabolism during developmental stages of C. elegans.

Biosynthesis and Site of Production of Sex Pheromone Components of the Cerambycid Beetle, Hedypathes betulinus

We determined the site of pheromone production tissues and a partial route for the biosynthesis of the sex pheromone in Hedypathes betulinus (Coleoptera: Cerambycidae: Lamiinae), Brazil's main green maté pest. Pheromone was found predominantly in the prothorax of males, suggesting that this is the region of production of pheromones in this insect. Scanning electron microscopy revealed small pores that may be associated with pheromone release in males; these pores also were observed in females. A deuterium-labeled putative precursor (geranyl acetone-D5) of the sex pheromone of H. betulinus was synthesized. When applied to the prothorax of males, label from the precursor was incorporated into the pheromone components, confirming that pheromone production occurs in the prothorax and that the pheromone components are biosynthesized from geranyl acetone.

CDNA CLONING AND SEQUENCE DETERMINATION OF THE PHEROMONE BIOSYNTHESIS ACTIVATING NEUROPEPTIDE FROM THE SEABUCKTHORN CARPENTERWORM, Holcocerus hippophaecolus (LEPIDOPTERA: COSSIDAE)

The PBAN (pheromone biosynthesis activating neuropeptide)/pyrokinin peptides comprise a major neuropeptide family characterized by a common FXPRL amide at the C-terminus. These peptides are actively involved in many essential endocrine functions. For the first time, we reported the cDNA cloning and sequence determination of the PBAN from the seabuckthorn carpenterworm, Holcocerus hippophaecolus, by using rapid amplification of cDNA ends. The full-length cDNA of Hh-DH-PBAN contained five peptides: diapause hormone (DH) homolog, α-neuropeptide (NP), β-NP, PBAN, and γ-NP. All of the peptides were amidated at their C-terminus and shared a conserved motif, FXPR (or K) L. Moreover, Hh-DH-PBAN had high homology to the other members of the PBAN peptide family: 56% with Manduca sexta, 66% with Bombyx mori, 77% with Helicoverpa zea, and 47% with Plutella xylostella. Phylogenetic analysis revealed that Hh-DH-PBAN was closely related to PBANs from Noctuidae, demonstrated by the relatively higher similarity compared with H. zea. In addition, real-time quantitative PCR (qRT-PCR) analysis showed that Hh-DH-PBAN mRNA expression peaked in the brain-subesophageal ganglion (Br-SOG) complex, and was also detected at high levels during larval and adult stages. The expression decreased significantly after pupation. These results provided information concerning molecular structure characteristics of Hh-DH-PBAN, whose expression profile suggested that the Hh-DH-PBAN gene might be correlated with larval development and sex pheromone biosynthesis in females of the H. hippophaecolus.

De Novo Biosynthesis of Sexual Pheromone in the Labial Gland of Bumblebee Males

De novo biosynthesis of male sex pheromone from two bumblebee species (Bombus terrestris and Bombus lucorum) was studied by using in vitro incubations of labial glands (LGs) with radioactive [1,2-(14)C]acetate and deuterated [D(3)]acetate. The labeled substrate was incorporated into several types of compounds, such as terpenic alcohols, fatty acids, esters, and hydrocarbons. A similar incubation of [1,2-(14)C]acetate with fat bodies (FB) led to the formation of fatty acids, triacylglycerols (TAG), and hydrocarbons. To support the results from in vitro incubations, PCR analysis of fatty acid synthase (FAS) transcripts in LG and FB was performed. Relative quantification of FAS transcription levels revealed that the abundance of mRNA from the FAS gene is a function of the age of B. terrestris males. A comparison of the relative FAS mRNA gene transcription level in FB and LGs of B. terrestris and B. lucorum males proved that high biosynthetic activity takes place in the LGs of both species. Together, these results indicate that pheromone components are synthesized de novo in the LG.

Molecular Mechanisms Underlying Insect Behaviors: Receptors, Peptides, and Biosynthetic Pathways

Molecular Mechanisms Underlying Insect Behaviors: Receptors, Peptides, and Biosynthetic Pathways

A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory

BackgroundMoths (Lepidoptera) are highly dependent on chemical communication to find a mate. Compared to conventional unselective insecticides, synthetic pheromones have successfully served to lure male moths as a specific and environmentally friendly way to control important pest species. However, the chemical synthesis and purification of the sex pheromone components in large amounts is a difficult and costly task. The repertoire of enzymes involved in moth pheromone biosynthesis in insecta can be seen as a library of specific catalysts that can be used to facilitate the synthesis of a particular chemical component. In this study, we present a novel approach to effectively aid in the preparation of semi-synthetic pheromone components using an engineered vector co-expressing two key biosynthetic enzymes in a simple yeast cell factory.ResultsWe first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum. The ∆11-desaturase produced predominantly Z11-16:acyl, a common pheromone component precursor, from the abundant yeast palmitic acid and the FAR transformed a series of saturated and unsaturated fatty acids into their corresponding alcohols which may serve as pheromone components in many moth species. Secondly, when we co-expressed the genes in the Brewer’s yeast Saccharomyces cerevisiae, a set of long-chain fatty acids and alcohols that are not naturally occurring in yeast were produced from inherent yeast fatty acids, and the presence of (Z)-11-hexadecenol (Z11-16:OH), demonstrated that both heterologous enzymes were active in concert. A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH. Finally, we demonstrated that oxidized extracts from the yeast cells containing (Z)-11-hexadecenal and other aldehyde pheromone compounds elicited specific electrophysiological activity from male antennae of the Tobacco budworm, Heliothis virescens, supporting the idea that genes from different species can be used as a molecular toolbox to produce pheromone components or pheromone component precursors of potential use for control of a variety of moths.ConclusionsThis study is a first proof-of-principle that it is possible to “brew” biologically active moth pheromone components through in vitro co-expression of pheromone biosynthetic enzymes, without having to provide supplementary precursors. Substrates present in the yeast alone appear to be sufficient.

Candidate chemosensory genes in the Stemborer Sesamia nonagrioides.

The stemborer Sesamia nonagrioides is an important pest of maize in the Mediterranean Basin. Like other moths, this noctuid uses its chemosensory system to efficiently interact with its environment. However, very little is known on the molecular mechanisms that underlie chemosensation in this species. Here, we used next-generation sequencing (454 and Illumina) on different tissues from adult and larvae, including chemosensory organs and female ovipositors, to describe the chemosensory transcriptome of S. nonagrioides and identify key molecular components of the pheromone production and detection systems. We identified a total of 68 candidate chemosensory genes in this species, including 31 candidate binding-proteins and 23 chemosensory receptors. In particular, we retrieved the three co-receptors Orco, IR25a and IR8a necessary for chemosensory receptor functioning. Focusing on the pheromonal communication system, we identified a new pheromone-binding protein in this species, four candidate pheromone receptors and 12 carboxylesterases as candidate acetate degrading enzymes. In addition, we identified enzymes putatively involved in S. nonagrioides pheromone biosynthesis, including a ∆11-desaturase and different acetyltransferases and reductases. RNAseq analyses and RT-PCR were combined to profile gene expression in different tissues. This study constitutes the first large scale description of chemosensory genes in S. nonagrioides.

Ant trail pheromone biosynthesis is triggered by a neuropeptide hormone.

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour's gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN's role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta.

Mimicking Insect Communication: Release and Detection of Pheromone, Biosynthesized by an Alcohol Acetyl Transferase Immobilized in a Microreactor

Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis, is achieved in a novel microfluidic system designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an “artificial gland”, i.e., a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behavior as 3 virgin females in most of the parameters considered.

Stylopsal: The First Identified Female-produced Sex Pheromone of Strepsiptera

A female-produced sex pheromone of Stylops muelleri was identified as an unusually branched saturated aldehyde (9R)-3,5-syn-3,5,9-trimethyldodecanal. We named it stylopsal. Its structure was established by using mass spectrometry, infrared spectroscopy, and organic synthesis of candidate compounds. The synthetic standard of (9R)-3,5-syn-3,5,9-trimethyldodecanal gave identical chromatographic and mass spectrometric data as the natural pheromone and also was active in electroantennographic and behavioral assays. The female fat body lipids contained the corresponding fatty acid, indicating a possible link between lipid metabolism and the pheromone biosynthesis.

Synthetic rates of key stored fatty acids in the biosynthesis of sex pheromone in the moth Heliothis virescens

Using a tracer–tracee approach, we fed 1-d-old virgin Heliothis virescens U-13C-glucose and analyzed the key labeled fatty acids, (Z)-11-hexadecenoate, hexadecanoate and octadecanoate, known to be intermediates in pheromone biosynthesis, by mass isotopomer distribution analysis. This method allowed determination of enrichment, and fractional (FSR) and absolute (ASR) synthetic rates. As expected, FSRs and ASRs for all three moieties were greater in the scotophase than photophase. However, in whole gland extracts, FSRs and ASRs of (Z)-11-hexadecenoate and hexadecanoate were much lower than those of the major pheromone component, (Z)-11-hexadecenal, determined previously. Since pheromone is made via these acids, we postulated that pheromone was produced directly and very rapidly via a small pool of acyl CoA thioesters of these acids and that the pool of acids we analyzed in our whole gland extract was largely a ‘dead end’ pool of excess acids (i.e., not converted directly to pheromone) stored in glycerolipids. We tested this by fractionating the whole glandular extract and analyzing the glycerolipid fraction. FSRs and ASRs for the two acids in the glycerolipid fraction were similar to those for the whole gland extract, confirming our postulate. Thus, most acetate produced in the pheromone gland is converted rapidly and directly to pheromone, while excess fatty acids are stored in glycerolipids and remain relatively inaccessible for pheromone production, at least over the two periods studied. Precursor enrichment of octadecanoate was substantially lower than that determined for the two 16-carbon acids and pheromone component. This suggests that hexadecanoate is the principal product of the multi-enzyme complex fatty acid synthase in the gland, and that octadecanoate is formed by subsequent chain elongation of hexadecanoate.

Targeted Metabolomics Reveals a Male Pheromone and Sex-Specific Ascaroside Biosynthesis in Caenorhabditis elegans

In the model organism Caenorhabditis elegans, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC-MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles.

Phenotypic impacts of PBAN RNA interference in an ant, Solenopsis invicta, and a moth, Helicoverpa zea

Insect neuropeptide hormones represent more than 90% of all insect hormones. The PBAN/pyrokinin family is a major group of insect neuropeptides, and they are expected to be found from all insect groups. These species-specific neuropeptides have been shown to have a variety of functions from embryo to adult. PBAN is well understood in moth species relative to sex pheromone biosynthesis, but other potential functions are yet to be determined. Recently, we focused on defining the PBAN gene and peptides in fire ants in preparation for an investigation of their function(s). RNA interference (RNAi) technology is a convenient tool to investigate unknown physiological functions in insects, and it is now an emerging method for development of novel biologically-based control agents as alternatives to insecticides. This could be a paradigm shift that will avoid many problems associated with conventional chemical insecticides. In this study, we selected the PBAN gene and its neuropeptide products as an RNAi target from two insect groups; a social insect, the fire ant (Solenopsis invicta) and a non-social insect, the corn earworm (Helicoverpa zea). Both insects are economically important pests. We report negative impacts after PBAN dsRNA treatment to suppress PBAN gene transcription during developmental and adult stages of both species, e.g. increased adult and larval mortality, delayed pupal development and decreased sex pheromone production in the moth. This is an important first step in determining the multiple functions of the PBAN gene in these two insects. This work illustrates the variety of phenotypic effects observed after RNAi silencing of the PBAN gene and suggests the possibility of novel biologically-based insect pest control methods.

Transcriptional analysis of sex pheromone biosynthesis signal genes in Bombyx mori

Abstract Six sex pheromone synthesis signal genes, including acyl coenzyme A (acyl‐CoA) desaturase (desat1), fatty acyl reductase (FAR), pheromone biosynthesis activating neuropeptide receptor (PBANR), fatty acid transport protein (FATP), acyl‐CoA binding protein (ACBP) and store‐operated channel protein (Orai1A), were studied for their transcriptional regulations. The expression profiles of these transcripts at different developmental stages (from –96 to 48 h) revealed that the genes are expressed in an age‐dependent manner. The transcripts of these genes continued to increase despite decapitation, and compared with normally developmental females, decapitation significantly inhibited their expression. Further experiments with a methoprene, a juvenile hormone (JH) analogue, challenge showed that JH was not a key inhibiting factor in the expression of these genes, and mating was found to significantly inhibit the expression of these marker genes. Altogether, the results provide a reference for understanding the mechanism of sex pheromone synthesis.

Sublethal effects of azadirachtin on lipid metabolism and sex pheromone biosynthesis of the Asian corn borer Ostrinia furnacalis

Azadirachtin has remarkable toxic and growth inhibitory effects on Ostrinia furnacalis (Guenee) (Lepidoptera: Pyralidae). In this study, sublethal effects of azadirachtin on fatty acid metabolism and sex pheromone biosynthesis in O. furnacalis were investigated. Quantities of fatty acids were significantly reduced when larvae were fed a diet containing azadirachtin at 0.1–10 ppm. After 10 days, fatty acids were reduced by 50% on a diet treated with 10 ppm azadirachtin; the relative composition of fatty acids was also affected. The relative proportion of C18:2 fatty acid decreased significantly in adult survivors, correlating with decreased fecundity. Furthermore, sex pheromone titers in adults were inversely proportional to dietary azadirachtin concentration, and reduced by 50% relative to controls when larvae were fed a diet containing 10 ppm azadirachtin. However, there were no effects of azadirachtin on pheromone blend ratios. Our results suggest that reductions in fecundity and pheromone titer in O. furnacalis are associated with alterations in lipid metabolism by azadirachtin, although these effects are not linked to a reduction in consumption.

Is the Sex Communication of Two Pyralid Moths, Plodia interpunctella and Ephestia kuehniella, under Circadian Clock Regulation?

Females of the Indian meal moth, Plodia interpunctella, and females of the Mediterranean flour month, Ephestia kuehniella (both Lepidoptera: Pyralidae), exhibit daily rhythms in calling behavior. The peak in P. interpunctella calling occurs at dusk, whereas E. kuehniella calls preferentially at dawn. This behavior turned arrhythmic in P. interpunctella females in constant darkness (DD) and remained arrhythmic in constant light (LL), whereas E. kuehniella females showed a persistent rhythm in DD and suppression of the behavior in LL, indicating regulation by a circadian clock mechanism. The rhythm of male locomotor activity corresponded well with the sexual activity of females, reaching the peak at dusk in P. interpunctella and at dawn in E. kuehniella. An immunohistochemical study of the pheromone biosynthesis activating neuropeptide, corazonin, and pigment dispersing factor revealed distinct sets of neurons in the brain-subesophageal complex and in the neurohemal organs of the 2 species.

Semi–Selective Fatty Acyl Reductases from Four Heliothine Moths Influence the Specific Pheromone Composition

BackgroundSex pheromones are essential in moth mate communication. Information on pheromone biosynthetic genes and enzymes is needed to comprehend the mechanisms that contribute to specificity of pheromone signals. Most heliothine moths use sex pheromones with (Z)–11–hexadecenal as the major component in combination with minor fatty aldehydes and alcohols. In this study we focus on four closely related species, Heliothis virescens, Heliothis subflexa, Helicoverpa armigera and Helicoverpa assulta, which use (Z)–11–hexadecenal, (Z)–9–tetradecanal, and (Z)–9–hexadecenal in different ratios in their pheromone blend. The components are produced from saturated fatty acid precursors by desaturation, β–oxidation, reduction and oxidation.ResultsWe analyzed the composition of fatty acyl pheromone precursors and correlated it to the pheromone composition. Next, we investigated whether the downstream fatty–acyl reduction step modulates the ratio of alcohol intermediates before the final oxidation step. By isolating and functionally characterizing the Fatty Acyl Reductase (pgFAR) from each species we found that the pgFARs were active on a broad set of C8 to C16 fatty acyl substrates including the key pheromone precursors, Z9–14, Z9–16 and Z11–16:acyls. When presenting the three precursors in equal ratios to yeast cultures expressing any of the four pgFARs, all reduced (Z)–9–tetradecenoate preferentially over (Z)–11–hexadecenoate, and the latter over (Z)–9–hexadecenoate. Finally, when manipulating the precursor ratios in vitro, we found that the pgFARs display small differences in the biochemical activity on various substrates.ConclusionsWe conclude that a pgFAR with broad specificity is involved in heliothine moth pheromone biosynthesis, functioning as a semi–selective funnel that produces species–specific alcohol product ratios depending on the fatty–acyl precursor ratio in the pheromone gland. This study further supports the key role of these in pheromone biosynthesis and emphasizes the interplay between the pheromone fatty acyl precursors and the Lepidoptera specific pgFARs in shaping the pheromone composition.